A Designer Peptide as a Template for Growing Au Nanoclusters ·  · 2014-08-07A Designer Peptide as a Template for Growing Au Nanoclusters ... S(q)= where N is the number of particles

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Supporting Information for:

A Designer Peptide as a Template for Growing Au Nanoclusters

Roberto de la Rica*, Lesley W. Chow, Christine-Maria Horejs, Manuel Mazo, Ciro Chiappini, E.

Thomas Pashuck, Ronit Bitton, Molly M. Stevens*

Contents:

- Figure S1: High-resolution TEM images of Au nanoclusters

- Figure S2: TEM images of Au nanoparticles grown without peptide

- Figure S3: TEM images of Au nanoparticles grown with (GRP)3

- Figure S4: Internalization of Au nanoclusters by cells

- Materials and methods

- References

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Figure S1. HRTEM image of Au nanoclusters grown with C(GRP)3.

Figure S2. TEM image of Au nanoparticles grown without peptide.

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Figure S3. TEM image of Au nanoparticle aggregates grown with the Cys-free peptide (GRP)3.

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Figure S4. Internalization of Au nanoclusters by human fibroblasts; (a) confocal microscopy

images showing cell membrane (green), nucleus (blue) and Au nanoclusters (red); nanoclusters are

located both in vesicles and free in the cytosol; Z axis scale bar: 2 mm; (b) cytotoxicity assay after

24 h in the presence of nanoclusters (green color shows live cells and red color shows dead cells);

(c) Metabolic activity (Alamar blue assay) of human fibroblasts after incubation with peptide-

templated nanoclusters at different concentrations calculated with respect to a sample with no

nanoclusters added. Analysis with a Mann-Whitney U test reveals statistical differences in

metabolic activity only when the peptide concentration is higher than 1 mg·mL-1. Error bars are the

standard deviation (n=3).

Materials and methods

Peptides were synthesized manually using standard Fmoc solid phase peptide synthesis

techniques. Rink amide resin, Fmoc-protected amino acids, N, N dimethyl formamide (DMF),

dichloromethane (DCM), 20% piperidine in DMF, O-Benzotriazole-N,N,N’,N’-

tetramethyluronium-hexafluoro-phosphate (HBTU), and diisopropylethylamine (DIEA) were

purchased from AGTC Bioproducts. All other solvents were purchased from Sigma. Peptides were

synthesized manually using standard Fmoc solid phase peptide synthesis techniques on a 0.5 mmole

scale. For each coupling, the Fmoc protecting group was removed with 20% piperidine in DMF

followed by washing with DCM and DMF. Amino acids were activated by adding 4 molar

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equivalents of the Fmoc protected amino acids to 3.95 molar equivalents of HBTU and dissolved in

DMF. Six molar equivalents of DIEA were added to the amino acid solution, and the coupling

solution was added to the resin. The coupling reaction was allowed to proceed for two to three

hours then the resin was washed in DCM and DMF. Ninhydrin tests were performed after each

Fmoc deprotection and coupling step to monitor the presence of free amines. Peptides were cleaved

in 95% trifluoroacetic acid (TFA), 2.5% triisopropyl silane (TIS) and 2.5% H2O for four hours.

TFA was removed using rotary evaporation, and the peptide residue was precipitated and washed

with cold diethyl ether by centrifugation. The peptide precipitate was allowed to dry under vacuum

to remove residual ether. The peptide was purified using reversed phase preparative high

performance liquid chromatography (HPLC; Shimadzu) in an acetonitrile/water gradient under

acidic conditions on a Phenomenex C18 Gemini NX column (5 micron pore size, a 110Å particle

size,150 x 21.2 mm). Following purification, the peptide was lyophilized on a freeze dryer

(Labconco) for storage prior to use. The purified peptide mass was verified by matrix assisted laser

desorption spectroscopy (MALDI; Waters).

To grow Au nanoparticles and Au nanoclusters, 5 mL of gold (III) chloride trihydrate (100

mM, Sigma) and 1.5 mL of the peptide (1 mg·mL-1 in water) were added to 3 mL of deionized

water under agitation (900 rpm). Subsequently, 500 mL of freshly prepared NaBH4 (10 mM) was

added to the stirred solution. The color of the solution changed instantaneously. Au nanocluster

solutions were lyophilized and stored at room temperature until used.

Molecular dynamic simulations were performed using the software NAMD. NAMD was

developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for

Advanced Science and Technology at the University of Illinois at Urbana-Champaign [1] and is free

to download and use at www.ks.uiuc.edu/Research/namd/. All visualizations of the simulation

results were made with the molecular graphics program VMD [2] which can be downloaded at

www.ks.uiuc.edu/Research/vmd/ and PyMOL Molecular Graphics System, Schrödinger, LLC.

NAMD simulation software generates structure files from the CHARMM force field, uses periodic

boundary conditions, and the particle-mesh Ewald method [3] PME for long-range interactions. The

equations of motion, that is, the time evolution of the Hamiltonian system, are integrated by the

Verlet method.[4] All simulations were computed at the SUN cluster Phoenix at

phoenix.zserv.tuwien.ac.at. Simulations were done in water using the TIP3P water model at

ambient conditions.

UV-Vis spectra were recorded with a Lambda 25 spectrometer (Perkin Elmer). Fluorescence

spectra were recorded with a Fluorolog fluorometer (Horiba). Polyacryalmide gel electrophoresis

was performed with a 15% acrylamide resolving gel topped with a 4% stacking gel. Gels were run

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in acidic native conditions. Bands were visualized by detecting the intrinsic NIR fluorescence of the

nanoclusters with a LI-COR infrared imaging system (Odyssey).

Small angle scattering measurements were performed using the SAXSLAB GANESHA

300-XL system with Cu Κα radiation generated by a sealed microfocused tube (Genix 3D Cu-

source with integrated Monochromator) powered at 50kV and 0.6mA, and three pinholes

collimation. The scattering patterns were recorded by a Pilatus 300K detector. The scattering

intensity I(q) was recorded in the interval 0.012 < q < 0.7 A-1 , where q is defined as 4 sinq πθ

λ=

where 2θ is the scattering angle, and λ is the radiation wavelength (1.542A). The solution under

study was sealed in a thin-walled capillary (glass) of about 1.5 mm diameter and 0.01 mm wall

thickness; measurements were performed at ambient conditions. The 2D SAXS images were

azimuthally averaged to produce one-dimensional profiles of intensity, I vs. q, using the two-

dimensional data reduction program SAXSGUI. The scattering spectra of the capillary and solvent

were also collected and subtracted from the corresponding solution data. No attempt was made to

convert the data to an absolute scale. Data analysis was based on fitting the scattering curve to an

appropriate model using a least-squares method using software provided by NIST (NIST SANS

analysis version 7.0 on IGOR).

SAXS analysis proceeded as follows. The scattering intensity of a monodisperse system of

particles of identical shape can be described as [5]:

I(q) NP(q)S(q)= where N is the number of particles per unit volume, S(q) is the structure factor that accounts for the

interparticle interactions and P(q) is the form factor characteristic of the specific size and shape of

the scatterers. In dilute solutions, where the interactions between the objects can be neglected, S(q)

equals one.

The total intensity scattered from a polydisperse system with particles having an identical shape can

be described by:

0

( ) ( ) ( , )nI q N D r P q r dr∞

= ∫

where Dn(r) is a distribution function and Dn(r)dr is the number of particles, the size of which is

between r and r + dr, per unit volume of sample.

A form factor for a sphere with radius r and a uniform electron density is given by [6]:

7

23

3

4 3(sin( ) cos( )3 ( )r qr qr qr

qrπ ρ⎡ ⎤Δ −

⋅⎢ ⎥⎣ ⎦

Where Δρ is the difference between the electron densities of the spheres and the solution.

A lognormal distribution is given by:

02

1( ) exp (ln( ) ln( ))22

drN r r rr σσ π

⎡ ⎤= − −⎢ ⎥⎣ ⎦

Where r0 is the median radius and the polydispersity is given by σ. The mean radius is given by: 2

0exp(ln( ) 2)avgr r σ= +

Human primary fibroblasts were kindly provided by Dr Felipe Prosper (Clinica Universidad

de Navarra, Pamplona, Spain). Briefly, cells were cultured in DMEM 4500 mg·L-1 glucose

supplemented with 10% fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v) antibiotics (all

from Invitrogen). Upon confluence, cells were detached with Trypsin/EDTA (Gibco) and plated at

10000 cells per well in 96 well plates. 24 hours later, growth medium was changed for nanoclusters

in serum-supplemented phenol-free DMEM (Invitrogen). After 24 hours, viable cells were

determined with a LIVE/DEAD assay (Invitrogen). Metabolic activity was determined with the

Alamar Blue assay (Invitrogen, see Supporting Information online). For confocal imaging, cells

were fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich), stained with Phalloidin-Alexafluor 488

(Invitrogen) and DAPI (Sigma-Aldrich) and mounted on a coverslip with prolong gold antifade

agent (Invitrogen). Micrographs were generated using an inverted confocal microscope (Leica SP5)

equipped with a 63x oil immersion objective with 1.4 NA. Images were processed using the

Volocity imaging software (Perkin Elmer).

References:

[1] J. C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot, R. D. Skeel,

L. Kalé, and K. Schulten, J. Comput.Chem. 2005, 26, 1782.

[2] W. Humphrey, A. Dalke, and K. Schulten, J. Mol. Graphics 1996, 14, 33.

[3] U. Essmann, L. Perera, M. L. Berkowitz, T. Darden, H. Lee, and L. G. Pedersen, J. Chem. Phys.

1995, 103, 8577.

[4] J. Verlet, Phys. Rev. 1967, 159, 98.

[5] O. Glatter, O. Kratky, Small angle x-ray scattering, Academic Press, London ; New York, 1982.

[6] J. S. Pedersen, Advances in Colloid and Interface Science, 1997, 70, 171-210.